Ionophoric Copper-Chelators in Combination with Mapk Inhibitors for Use in Treatment of Cancer

This application claims the right of priority of European Patent Application EP22178503.3 filed 10 Jun. 2022, which is incorporated by reference herein.

The present invention relates to the use of neocuproine, elesclomol, disulfiram and/or dithiocarbamate for use in treatment of cancer, optionally in combination with one or more MAPK-pathway inhibitors in cancers carrying mutations that are recognized as MAPK-pathway activating mutations.

Despite numerous powerful and successful studies on cancer, the rapid increase in cancer prevalence and cancer mortality rates is one of the biggest problems worldwide. The causes for these phenomena include environmental pollution, population growth and aging. In both genders the most lethal cancer types among solid tumors are lung cancer (with 14% mortality rate of the total cases) being the most prominent reason for death, followed by female breast cancer (13.6%), colon cancer (9%), liver cancer (8.2%) and prostate cancer (7.7%) (www.gco.iarc.fr). In most cancer patients, chemotherapy or radiotherapy are the primary approaches, together with excision of solid and local tumors. However, in the last two decades, cancer research has led to the development of small molecule therapies targeting activated kinases and -pathways. This is due to massive sequencing of cancer tissues revealing druggable common oncogenic driver mutations. Mutations in RAS oncogene (HRAS, NRAS, and KRAS) are among the most common ones. RAS is a small GTPase which is a regulator for important cellular functions such as proliferation, cell migration, programmed cell death (apoptosis) and survival. These cellular events need to be controlled tightly, and dysregulation caused by activating mutations can hyper-activate downstream signaling cascades and lead to malignant transformation, tumor growth and metastasis. For example, activating mutation in RAS, mainly involving amino acids G12, G13 or Q61, which lead to an over-activated RAS kinase, will hyper-activate the downstream signaling cascade called MAPK (mitogen-activated protein kinase) pathway, which normally controls expression of genes involved in proliferation, migration, invasion and cell fate determination. In contrast to other oncogenic activating mutations, there is no kinase inhibitor that directly targets RAS kinase. Therefore, downstream kinase inhibitors like panRAF (e.g. Belvarafenib, Naporafenib) or MEK inhibitors (e.g. Binimetinib, Trametinib etc) are used in such cases. Furthermore, mutations in the BRAF kinase or receptor-tyrosine kinases like cKit (v-kit proto-oncogen) or EGFR (epidermal-growth factor receptor) also lead to activated MAPK signaling. Here, direct kinase inhibitors are available (e.g. Dabrafenib, Encorafenib, Imatinib, Sunitinib). Despite extensive clinical research for the use of kinase inhibitors for solid tumors, there is still a lack of outstanding clinical successes, especially for MAPK-pathway inhibitor (MEKi, BRAFi, panRAFi) or tyrosine kinase inhibitor monotherapy, and relapse is mostly observed. Hence, a high medical need exists for novel combinatorial treatments that can increase the efficiency of kinase inhibitors.

Copper (Cu) is an essential trace element that is indispensable for life. This metal serves as catalytic and structural cofactor for enzymes involved in many physiological processes such as energy generation, iron acquisition, oxygen transport, cellular metabolism, signal transduction, and blood clotting. The homeostatic balance of bioavailable copper in the human body has shown to effect tumor growth and copper levels are elevated in cancer patients. Brady et al. showed that copper is a coactivator of the MAPK pathway by interacting with MEK1/2, enabling the phosphorylation MAPK down-stream target Erk. Moreover, the role of Ctrl, the transporter responsible for copper ion-influx into the cells was studied by interrupting its expression, which lead to decreased oncogenic BRAF signaling, thus highlighting the role of copper in MAPK-driven melanoma cells. Copper chelators used in the treatment of Wilsons's disease were suggested to be beneficial for cancer patients (Trientin). It was also shown that copper availability affects the growth of pancreatic tumors, and that intracellular copper uptake regulates a cancer cell metabolic phenotype shown in the presence of the copper chelator tetrathiomolybdate.

On the other hand, and in contrast to Cu chelators, the chemical class of Cu ionophores chelate metal ions in the extracellular space and transport them through biological membranes thus increasing the intracellular copper levels. In the case of Cu binding for example the reduction of bound Cu(II) to Cu(I) and the release of Cu ions from the ionophore will lead to the production of reactive-oxygen species (ROS), toxic to cancer cells. Copper ionophores are Cu dependent and in combination with Cu chelators (e.g. Trientin or tetrathiomolybdate) lose or decrease their toxic activity on cancer cells. Some copper chelators have been already studied extensively also for their clinical relevance. The most well-known are: Disulfiram and dithiocarbamates; clioquinol and hydroxyquinolines; elesclomol, and neocuproine.

Neocuproine, also known as 2,9-dimethyl-1,10-phenanthroline, is a member of the family of phenantrolines, and a specific Cu(I) chelator. Neocuproine is known as a copper chelating agent which upon binding of Cu(II) rapidly reduced to Neocuproine-Cu(I) complexes. Although these complexes are described as stable, it was observed that Neocuproine-Cu(I), in orchestra with the antioxidant-defense molecule Glutathione, induces DNA scission through oxidative mechanisms.

When neocuproine is given in solution together with e.g. CuSO, Cu(II)-neocuproine complexes are formed and it was noticed that there is synergistic cytotoxic effects when CuSOis added together with neocuproine on L1210 mouse lymphocytic leukemia cells. Neocuproine complexed with Cu(I) did show tumor promoting effects in a B16 mouse melanoma model, and also resulted in enhanced tumor pigmentation. On the other hand, when neocuproine was added into the water of adult zebrafish, it was shown to decrease pigmentation and to induce melanocytes death. Byrnes et al. showed that neocuproine inhibited the growth of Ehrlich ascites tumor cells and noticed the synergistic effect in inhibition of cellular growth when neocuproine was given together with CuCl. The cytotoxic and DNA damaging cellular effects is thought to be induced by hydroxyl radicals (oxidative stress) which are formed during the reduction of Copper to Cu(I) when bound to neocuproine while internalized into the cells. This effect was also demonstrated inwhere a synergistic effect of neocuproine with hydrogen peroxide resulted in increased number of DNA strand breaks and oxidative stress. Moreover, Cu(I) was suggested to be important for proteasome function, and it was shown that the presence of Neocuproine impairs that function, so it can be said that Neocuproine induces proteasome inhibition, which might be another mechanism of action, independent of its ROS inducing function, and which could inhibit cancer cells viability.

Elesclomol is a bis(thiohydrazide) amide, and similar to neocuproine, a compound that binds copper. Elesclomol was identified through an original cell-based multidrug resistance modulators screen where it was synthesised from a given parental compound library (Chen, S., et al., Bioorg Med Chem Lett, 2013. 23(18): p. 5070-6). It was further developed by Synta Pharmaceuticals and previously tested in several clinical trials in a combination treatment with paclitaxel for anticancer activity of solid tumours including metastatic melanoma (Berkenblit, A., et al., Clin Cancer Res, 2007. 13(2 Pt 1): p. 584-90; O'Day, S., et al., J Clin Oncol, 2009. 27(32): p. 5452-8, O'Day, S. J., et al., J Clin Oncol, 2013. 31(9): p. 1211-8.). Mechanistically, elesclomol binds Cu(II) in the extracellular environment and forms a membrane permeable complex, which upon entering the mitochondria releases copper after it is reduced to Cu(I). Cu(I) released in the mitochondria can react with molecular oxygen to generate ROS, which can cause unmitigated oxidative stress and apoptotic death of cancer cells.

DSF is an orally administered drug for the treatment of alcoholism. The drug inhibits the enzyme aldehyde dehydrogenase, which is an enzyme important for the alcohol metabolism in the liver. The combination of DSF and alcohol therefore leads to alcohol intolerance.

Another chemical function of DSF is the conversion to DDC in the stomach, which creates copper-DDC complexes. These complexes are known to increase the intracellular Cu levels and therefore DDC is classified as an ionophoric copper-chelator. Also, its intracellular action is abolished when given in combination with non-permeable Cu-chelators (e.g. Trientine). There are several clinical studies ongoing that investigate DSF for the treatment of cancer, but none of them is suggesting the combination of disulfiram in combination with binimetinib for the treatment of metastatic melanoma (Babak, M. V. et al., Biomedicines, 2021. 9(8)).

ROS (reactive oxygen species) is a collective term used to describe chemical species that are produced as byproducts of normal oxygen metabolism and include superoxide anion (O2-), hydrogen peroxide (HO), and the hydroxyl radical. Upregulation of ROS is often associated with chemotherapy or kinase inhibitor treatment of tumor cells. For example, it was demonstrated that oncogenic BRAF mutations, resulting in hyper-activation of MAPK signaling, maintain a glycolytic phenotype in melanoma, thus delivering anti-oxidant defense mechanism via the pentose phosphate pathway to insure survival of cancer cells and linking glycolysis with intracellular ROS levels. In contrast to the Warburg hypothesis, according to which tumors produce large portion of energy (ATP) through glycolysis, studies have shown that melanomas, when treated with MAPK pathways inhibitors, where glycolysis is decreased, have elevated level of oxygen consumption and therefore high oxidative phosphorylation rates (OXPHOS), resulting in elevated beta-oxidation and altered generation of superoxide anions. Moreover, metastasizing cells have intrinsic high ROS levels due to low glycolysis turnover and these cells are highly depending on NADPH-detoxifying enzymes. Any misbalance of NADPH recycling enzymes or other interventions, which result in elevated ROS levels, significantly decrease metastasis. In colorectal cancer resistance to chemotherapies leading to a transient diapause-like dedifferentiated cell state, which is maintained by upregulation of autophagy and decreased transcription but display high intracellular ROS levels. Non-small cell lung cancer (NSCLC) tumors implicate heterogeneity within cancer cell populations as a response to drug treatments. Drug-tolerant persister cells survive treatment with inhibitor, but are slow cycling. These upregulate insulin growth factor (IGF) signaling and alter chromatin state by histone demethylase activity. As a result, intracellular ROS levels are upregulated. In breast cancer, resistance to the tyrosine kinase inhibitor lapatinib is achieved through metabolic adaptation favoring mitochondrial energy metabolism through increased glutamine metabolism, resulting in ROS production. Moreover, targeting enhanced oxidative phosphorylation (OXPHOS), where elevated ROS is seen as a by-product, inhibits bone metastasis in triple-negative breast cancer (TNBC) cells.

In non-cancer cells, ROS are produced at low concentration and therefore effectively neutralized by the potent antioxidant system of the cells. Therefore, targeting ROS in aggressive cancer cells with already elevated ROS levels could be a promising treatment option). In summary, the combination of MAPK inhibitors, which have been shown to target tumor growth and ionophoric copper chelators, which induce ROS effectively in cancer cells, have a beneficial effect on tumor cell growth, metastasis and apoptosis.

In one aspect, the invention relates to the use of neocuproine in treatment of cancer characterized by cells having an oncogenic mutation associated with significantly increased occurrence of malignant tumours, selected from the group consisting of: an NRAS mutation, a KRAS mutation, an HRAS mutation, a BRAF mutation, a cKIT mutation, an NF1 loss of function mutation.

In another aspect, the invention relates to a combination medicament comprising neocuproine and a MAPK-pathway inhibitor. This combination medicament may be used in treatment of cancer.

A further aspect of the invention relates to a combination medicament comprising neocuproine and a tyrosine kinase inhibitor. This combination medicament may be used in treatment of cancer.

In yet another aspect, the invention relates to the use of a compound selected from the group comprising neocuproine, elesclomol, disulfiram, and dithiocarbamate for use in treatment of cancer.

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

The terms “comprising,” “having,” “containing,” and “including,” and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

As used herein, the term pharmaceutical composition refers to a compound of the invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration.

As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624).

As used herein, the term treating or treatment of any disease or disorder (e.g. cancer) refers in one embodiment to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and/or prevention of disease are generally known in the art, unless specifically described hereinbelow.

In the context of the present specification, the term resistant to treatment shall mean that the cells that are thus characterized do not respond to the indicated treatment.

In the context of the present specification, the term refractory to treatment shall mean that the cells that are thus characterized do not respond as well to the indicated treatment as to achieve the desired therapeutic effect expected for fully responsive cells.

In the context of the present specification, the term gene refers to the DNA sequence encoding a particular protein. When the term mutation refers to a protein name, it is understood that the respective DNA sequence harbours the mutation which leads to a change in the amino acid sequence of the protein.

In the context of the present specification, the term mutation refers to a non-silent change in the nucleic acid sequence of a gene. In certain embodiments, this change is a substitution of one or more base pairs.

In the context of the present specification, the term constitutively activating refers to an enzyme which is active even without activation of the upstream signalling cascade.

In the context of the present specification, the term loss of function refers to a protein which is not able to fulfil its native function to a similar extend as the wildtype gene.

The abbreviations of genes are listed below with their identifiers:

In recent years, targeting the MAPK-pathway has shown dramatic clinical effects. In melanoma for example, combined MAPK-pathway inhibitor therapy (BRAF inhibition+MEK inhibition), led to improvements in 5-year overall survival in patients with BRAF mutated tumours. Due to the lack of durably efficient targeted therapies against mutated RAS kinase proteins, immunotherapies are the recommended first-line treatment for this patient cohort. MEK inhibitor monotherapy in NRAS mutated melanoma can prolong progression-free survival and was suggested as a treatment option after the failure of immunotherapy. Unfortunately, targeting MAPK-signaling in NRAS-mutated melanoma is only beneficial to a small subset of patients (response rate for Binimetinib is 15-20%). Moreover, combined MAPK-pathway inhibition frequently results in resistance formation. One major obstacle in the treatment of solid tumors in general is the intra-tumor heterogeneity, which is derived from transcriptional cell plasticity (also called by the term epithelial-mesenchymal transition).

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods for the therapy of cancers with activation of MAPK signalling. This objective is attained by the claims of the present specification.

The present invention focuses on the treatment of tumor cell heterogeneity by combining inhibitors of the MAPK signaling pathway (MAPK-pathway) or tyrosine kinase inhibitors and small molecules which are able to induce ROS, known to be toxic for resistant cancer cells especially. The inventors show that the combination of a MAPK inhibitor or tyrosine kinase inhibitors and a ROS inducer lead to greater tumor growth inhibition and also can prolong the survival in vivo when compared to MAPK inhibition or tyrosine kinase inhibition alone.

A first aspect of the invention relates to neocuproine for use in treatment of cancer.

An alternative of the first aspect of the invention relates to elesclomol for use in treatment of cancer.

An alternative of the first aspect of the invention relates to disulfiram for use in treatment of cancer.

An alternative of the first aspect of the invention relates to dithiocarbamate for use in treatment of cancer.

In certain embodiments, the cancer is characterized by cells having a constitutively activating NRAS mutation. In certain embodiments, the NRAS mutation is selected from the group comprising NRAS Q61K, NRAS Q61L, NRAS Q61R, NRAS Q61H, or NRAS G12A.

In certain embodiments, the NRAS mutation is selected from the group of an NRAS Q61 mutation, an NRAS G12 mutation and an NRAS G13 mutation. In certain embodiments, the NRAS mutation is selected from the group of NRAS Q61R, NRAS Q61K, NRAS Q61L, and NRAS Q61H, NRAS G12D, NRAS G12S, NRAS G12D, NRAS G12C, NRAS G12V, NRAS G12A, NRAS G13D, NRAS G13R, NRAS G13V, NRAS G13C. In certain embodiments, the NRAS mutation is selected from the group of NRAS Q61K, NRAS Q61L, NRAS Q61R, NRAS Q61H, and NRAS G12A.

In certain embodiments, the cancer is characterized by cells having a constitutively activating KRAS mutation.

In certain embodiments, the KRAS mutation is selected from the group of a KRAS Q61 mutation, a KRAS G12 mutation, and a KRAS G13 mutation. In certain embodiments, the KRAS mutation is selected from the group of KRAS Q61R, KRAS Q61K, KRAS Q61 L, KRAS Q61H, KRAS G13D, KRAS G13R, KRAS G13V, KRAS G13C, KRAS G12D, KRAS G12S, KRAS G12D, KRAS G12C, KRAS G12V, and KRAS G12A.

In certain embodiments, the cancer is characterized by cells having a constitutively activating HRAS mutation.

In certain embodiments, the HRAS mutation is selected from the group of an HRAS Q61 mutation, an HRAS G12 mutation, and an HRAS G13 mutation. In certain embodiments, the HRAS mutation is selected from the group of HRAS Q61R, HRAS Q61K, HRAS Q61 L, HRAS Q61H, HRAS G12D, HRAS G12S, HRAS G12D, HRAS G12C, HRAS G12V, HRAS G12A, HRAS G13D, HRAS G13R, HRAS G13V, and HRAS G13C.

In certain embodiments, the cancer is characterized by cells having a constitutively activating BRAF mutation. In certain embodiments, the BRAF mutation is selected from the group comprising BRAF V600E, BRAF V600K, BRAF V600D or BRAF V600R.

In certain embodiments, the cancer is characterized by cells having a constitutively activating cKIT mutation. In certain embodiments, the cKIT mutation is selected from the group comprising cKIT K642E, cKIT L576P, cKIT V559A and cKIT W557R.

In certain embodiments, the cancer is characterized by cells characterized by a mutation in two genes of the group comprising NRAS, BRAF, and cKIT. In certain embodiments, the cancer is characterized by cells characterized by a mutation in NRAS, and BRAF. In certain embodiments, the cancer is characterized by cells characterized by a mutation in NRAS, and cKIT. In certain embodiments, the cancer is characterized by cells characterized by a mutation in BRAF and cKIT.

In certain embodiments, the said cancer is characterized by cells characterized by a mutation in all three genes of the group comprising NRAS, BRAF, and cKIT.

In certain embodiments, the cancer is characterized by cells that are resistant or refractory to treatment with a MEK inhibitor. In certain embodiments, the MEK inhibitor is selected from the group consisting of trametinib, binimetinib, cobimetinib and selumetninib.